PROJECT SUMMARY/ABSTRACT Preoperative or neoadjuvant therapy (NT) is increasingly used in patients with locally advanced or inflammatory breast cancer (BC) to allow optimal surgery. Although a pathologic complete response has been associated with increased survival, many patients do not respond and/or develop lethal metastatic disease after NT. Our preliminary data indicate that chemotherapy induces blood levels of monocyte chemoattractant proteins (MCPs), which can stimulate the cancer stem-like cell (CSC) phenotype to promote tumor malignancy. The goal of this study is to dissect the mechanisms through which NT regimens induce the CSC phenotype in breast tumors. We will investigate both a MCP-mediated systemic mechanism and a local mechanism mediated by cancer-secreted miRNAs, and will test intervention strategies that block these events during chemotherapy. The overall goal is to design interventions that maximize the beneficial effects of anticancer treatment by preventing NT-induced CSC expansion. In Aim 1, we will first use human and mouse BC cells to determine how receptor activation by MCPs leads to Numb degradation and Notch activation to promote CSCs. Additional effectors mediating MCPs' effect on cancer cells will be identified. Mouse tumor models will be used to determine the effect of MCPs on non-cancer cells in the tumor microenvironment, which may in turn regulate the cytokine environment to influence CSCs. In Aim 2, we will determine if the chemotherapy-induced miRNAs identified in our preliminary study synergistically stimulate CSCs with systemically elevated MCPs. In Aim 3, patient-derived xenograft tumor models and mouse tumor models will be used to determine the anti-CSC effects of various agents targeting the herein identified pathways. We will then determine if NT induces monocyte expansion in BC patients and if MCP-initiated signaling is associated with CSC frequency in primary human BCs. Results from the proposed work will provide a mechanistic and pre-clinical basis for a future clinical trial using one of the CCR2 inhibitors previously developed for non-cancer diseases to target treatment-induced CSCs in BC patients. Improving our understanding of the interplay between hematopoietic cells, bulk cancer cells, and CSC populations after NT will allow the development of improved combinatorial therapies to reduce therapeutic resistance and tumor relapse. It may also provide insight into the clinical application of therapeutic regimens tailored to the need of individual patients, ultimately leading to an increased success of anticancer treatment.